The increasing incidence of infections by the intestinal anaerobe Clostridium difficile coupled with the spread of drug resistant highly virulent strains dictates the need for developing new therapies for this potentially lethal pathogen. The goal of this project is to develop a novel therapy against C. difficile based on a combination of the well-tolerated antimicrobial berberine and an inhibitor of Multidrug Efflux Pumps (MDRs). Berberine is the principal component of Hydrastis canadensis (Goldenseal), and our previous research determined that its efficacy against bacterial pathogens is limited by MDRs of the MF family. However, MDR inhibitors increase the activity of berberine against gram positive bacteria, including C. difficile, by more than 60 fold. Similar results were found for a conjugate of berberine and an MDR inhibitor. Since the only known mechanism of resistance to berberine is MDR efflux, its combination with an MDR pump inhibitor, either separately or as a hybrid molecule, can result in a powerful antimicrobial. Importantly, the berberine/MDR inhibitor combination had strong bactericidal activity against stationary cells of C. difficile, in contrast to commonly used metronidazole and vancomycin, which were only active against growing bacteria. Upon reaching stationary state, C. difficile produces spores, which are responsible for the relapse of the infection. By killing stationary cells, berberine/MDR inhibitor combination prevents spore formation. This suggests that the combination antimicrobial may have a critical advantage over existing therapeutics by preventing relapse. Inhibitors against MF MDRs are found with high probability in compound libraries (~5%), presenting a unique opportunity to pick and choose leads with attractive properties (including poor absorption). The very high probability of finding MDR inhibitors provides a unique opportunity to rationally manage drug resistance. Conventional antimicrobials eventually fail due to resistance development. The advantage of our approach is that new classes of MDR inhibitors can be relatively easily developed from an enormous base of hit compounds. In this manner, we will be able to stay ahead of pathogen resistance. While finding MDR inhibitor hits has a high probability, developing them into leads presents a challenge as well, since it has been impractical to test a large number of compounds for toxicity and efficacy in vivo. We have recently described a whole-animal screen in C. elegans, that helps overcome these bottlenecks in development. We will use this model to rapidly identify attractive leads which will feed our drug development pipeline. Leads, including those we already identified, will be evaluated in vitro (potency, spectrum, cytotoxicity, absorbance) and then in mice for toxicity, and in a hamster model of C. difficile infection for efficacy. This multidisciplinary project is a collaboration between experts in antimicrobial chemotherapy, medicinal chemistry, C. difficile biology, host-pathogen interactions and veterinary medicine who have worked together on preliminary stages of this project.